Let-7d lentiviral vector induces the differentiation of rat bone marrow mesenchymal stem cells into neurons in vitro

doi:10.3969/j.issn.2095-4344.2014.41.008

Abstract

Abstract:

BACKGROUND: MicroRNA plays an important role in the process of growth and aging of living body. To know the role of let-7d in inducing bone marrow mesenchymal stem cell differentiation into neurons can promote the stem cell transplantation.

OBJECTIVE: To investigate the role of let-7d in inducing bone marrow mesenchymal stem cell differentiation into neurons.

METHODS: (1) The lentiviral vector of let-7d was constructed and transfected into rat bone marrow mesenchymal stem cells. The cells were divided into non-transfected group, negative control group (transfected with FU-RNAi-NC-LV), transfected enhancement group (transfected with let-7d-LV), transfected inhibition group ( transfected with let-7d-inhibition-LV). (2) Rat bone marrow mesenchymal stem cells were treated with fasudil as an inducer for triggering the cells to differentiate into neurons. The expression of neuron-specific markers, neuron-specific enolase and microtubule-associated protein 2, were measured by immunocytochemical method. The mRNA expression of microtubule-associated protein 2 was detected by RT-PCR. The viability of bone marrow mesenchymal stem cells was determined by MTT method.
RESULTS AND CONCLUSION: Under inverted fluorescence microscope, the cells were successfully transfected with let-7d. Fasudil induced bone marrow mesenchymal stem cells to differentiate into neurons. The transfection efficiency and expression levels of neuron-specific enolase and microtubule-associated protein 2 in transfected enhancement group were higher than those in the negative control group (P < 0.05); while in the inhibition group, they were lower than those in the negative control group (P < 0.05). These findings indicate that let-7d can promote the differentiation of bone marrow mesenchymal stem cells into neurons induced by fasudil, and by controlling the expression of let-7d we can influence the differentiation efficiency from bone marrow mesenchymal stem cells to neurons.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, lentivirus, neurons

CLC Number:

R394.2

Xu Xiao-ge, Zhang Jing, Gong Zhe, Zhao Shao-yun, He Xia, Wang Tian-shu, Jiao Shu-jie, Teng Jun-fang, Jia Yan-jie. Let-7d lentiviral vector induces the differentiation of rat bone marrow mesenchymal stem cells into neurons in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6603-6608.

Figures/Tables 2

2.1 let-7d过表达和抑制慢病毒载体的构建 pFU-GW- iRNA载体经测序结果证明大鼠let-7d慢病毒载体构建成功。经293T细胞包装后，let-7d-LV滴度为6×108 TU/mL，let-7d-inhibition-LV滴度为3×108 TU/mL，空白病毒滴度为8×108 TU/mL。 2.2 let-7d慢病毒载体感染情况倒置荧光显微镜观察发现，慢病毒载体感染骨髓间充质干细胞2 d后，部分细胞脱落死亡，而且转染5 d时慢病毒载体自身携带的绿色荧光蛋白表达最为理想，随时间延长，荧光减弱(图1)，转染慢病毒载体的各组荧光表达无明显变化。 2.3 MTT法观察细胞存活率转染前各组骨髓间充质干细胞的MTT结果比较差异无显著性意义(P > 0.05)；转染24 h，转染组细胞存活率显著下降，与未转染组细胞存活率比较差异均有显著性意义(P < 0.05)，转染各组之间相比细胞存活率差异无显著性意义(P > 0.05)。转染各组转染120 h后细胞存活率虽较24 h有所上升(P < 0.05)，但与未转染组细胞存活率比较差异仍有显著性意义(P < 0.05)，见表1。 2.4 法舒地尔体外诱导骨髓间充质干细胞分化为神经细胞情况未转染组骨髓间充质干细胞诱导3 h，部分细胞出现神经细胞改变，胞体收缩成圆形，突起细长，有多个分支，部分在局部形成网络。转染上调组诱导后，细胞形态变化更为迅速和显著，诱导3 h后多数细胞胞体收缩成圆形，细胞突起细长，交织成网，形成较典型的神经细胞结构。感染下调组诱导后，细胞形态变化更为缓慢，诱导3 h后少量细胞出现神经细胞改变，胞体收缩呈圆形，未见网络形成(图2)。 2.5 免疫细胞化学染色法观察神经元标记物转染上调组诱导3 h后，NSE和MAP-2蛋白表达率分别为(92.1± 1.5)%、(89.4±2.5)%，显著高于未转染组[(65.3±1.2)%，(63.7±2.3%)]和阴性对照组[(64.7±2.1)%，(62.9±1.7%)] (P < 0.05)。转染下调组诱导3 h后，NSE和MAP-2蛋白表达率分别为(49.0±1.2)%、(47.0±1.6)%，显著低于未转染组和阴性对照组(P < 0.05)，见图3。 "

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